The detection of substances in various biological fluids by immunoassay methods is well known and used frequently for a variety of different purposes. Examples include the detection of antibodies in blood, urine, saliva or other biological fluids as an indication of the presence of a pathogen for diagnosis of various diseases and conditions. Other immunoassays of biological fluids include pregnancy tests and tests to determine blood alcohol level. While such tests can be carried out as liquid assays, it is often easier and more convenient to spot the sample onto a solid substrate on which a ligand for the target molecule is immobilized and detect the presence of a specific binding complex. The most widespread immunoassay solid phase format used today is the enzyme-linked immunosorbent assay (ELISA).
An ELISA apparatus typically comprises a 96 well microtiter plate, the inside surfaces of which are coated with a ligand specific for a target molecule present in a sample. This binding or attachment of the ligand to the solid phase is not a chemical reaction but rather is believed to result from a physical or noncovalent interaction between the polystyrene matrix of the microtiter plate and the antigen. A sample suspected of containing the target molecule is placed in contact with the microtiter plate so that binding will occur between the ligand and any target molecule in the sample. Any unbound target molecules are then removed from the plate wells by several washing steps. A second ligand which specifically recognizes the target molecule and is linked to a signal-generating enzyme is then added. Detection of the enzyme which is indicative of the presence of the target molecule in the sample is typically performed by addition of reagents which produce a color change.
However, ligands used in immunoassays such as ELISAs are oftentimes composed of bacterial and viral lysate antigenic materials. Crude lysates have a disadvantage in that large amounts of pathogenic microbes have to be cultured in order to obtain the required material. Further, the antigenic material of related microorganisms often cause false positives because of cross-reactivity of target molecules with the related organisms.
Accordingly, recombinant proteins are also now being used for assay development. Recombinant proteins have an advantage in that large amounts of the protein can be produced by a host cell without other proteins from the pathogenic organism being present. Thus, it is possible to obtain a much purer ligand. However, it has been found that even recombinant proteins can be prone to false positive reactivity due to the limited amount of epitopes as compared to immunologically non-relevant material on the recombinant protein. Synthetic peptides are also being used in an attempt to maximize the specificity of immunoassays. A disadvantage of synthetic peptides, however, can be lower sensitivity.
Further, performance of an ELISA can be quite time consuming. For example, an established method for screening for the AIDS virus is to first carry out an ELISA, followed by confirmation of positives by Western Blot. Generally, the ELISA takes about 4 hours and the Western Blot, which includes an overnight incubation period, requires about 20 hours. While this method may be adequate for routine screening of blood samples, it is not adequate for screening in organ transplant situations wherein results are required prior to the maximum ischemic time for the organ.
A similar technology, referred to as enzyme-linked immunofiltration assay (ELIFA) , has been developed more recently in an attempt to overcome problems with false positive results and low sensitivity associated with ELISAs. ELIFAs function very similarly to ELISAs with the exception that ELIFA takes advantage of filtering the initial solution containing the ligand through a nitrocellulose membrane to bind it to the membrane. This filtering process facilitates "immunoconcentration" in that much higher levels of ligand bind to the membrane as compared to levels of ligand that bind to the surface of a microtiter plate. Target molecules in a sample are then bound to the ligand by incubation as in the ELISA method. However, any unbound target molecule is removed from the membrane by filtration of the unbound molecules through the membrane into a waste chamber. Bound molecule is detected by precipitating a colored product on the membrane.
This type of porous solid substrate is, in theory, very useful since it permits removal of the bulk of the sample from the substrate while the target molecule remains at the surface bound to the immobilized ligand. However, in practice there are considerable difficulties due to slow flow of the sample through the substrate thus making this type of assay also very time consuming.
Accordingly, several alternative solid substrate-based immunoassays and methodologies for conducting more rapid and/or reliable immunoassays have been developed.
For example, in PCT Application WO 95/26504 an assay involving the detection of a specific binding complex between a target molecule and ligand immobilized on a porous solid substrate is described wherein reliability of the assay is improved simply by wiping the surface of the substrate after the sample has been added.
Hiraoka et al. (U.S. Pat. No. 5,569,589) disclose a device for immunoassay comprising a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of an enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting the non-diffusible material. Thus, in this assay a target molecule is quantitatively analyzed by determining the change in enzymatic activity caused by a reaction between the target molecule, a linked product of the target molecule with a high molecular weight compound and an enzyme labeled antibody.
Ashihara et al. (U.S. Pat. No. 5,603,898) disclose a similar dry-type analytical device for immunoassay. This device contains at least one water permeable layer for measuring a target molecule in a sample according to enzyme immunoassay which comprises a water soluble macromolecular substrate and an enzyme-antibody conjugate of an enzyme capable of action on the water-soluble macromolecular substrate with an antibody reacting with the ligand in the sample.
Kang et al. (U.S. Pat. No. 5,559,041) disclose various embodiments of an immunoassay device comprising carbon black and ligands which are coupled to the carbon black. In one embodiment, a labeled reagent operable to produce a specific ligand-target molecule complex is uniformly impregnated through a first filter element. As a liquid sample emerges from a reservoir pad, it comes into contact with the reagent in the first filter element where it will react to form the specific ligand-target molecule complex. The sample then migrates through a second filter element located adjacent to the first filter element and distal to the reservoir pad which impedes passage of larger components contained therein but is operable to permit passage of any specific ligand-target molecule complex onto a wicking membrane. Labeled target molecule, if present, will bind to an assay indicia zone located in the wicking membrane to produce a visible signal.
WO 96/15453 describes a process for identifying and quantitatively determining the amount of a selected protein in a sample by passing the sample through a series of laminated membranes considered as two separate membrane units. The first membrane unit comprises a separator membrane superimposed in capillary contact with a reactor membrane. The separator membrane of the first unit is a microporous, hydrophilic asymmetric membrane, the average pore size of which decreases from top to bottom. The reactor membrane of the first unit functions to remove contaminating substances such as proteins which will interfere with the measurement to be performed. This is accomplished by immobilizing on the reactor membrane a sufficient number of antibodies so that substantially all the contaminating materials react with them, thereby becoming immobilized or irreversibly bound to the reactor membrane. The second membrane unit comprises a collector membrane superimposed and in capillary contact with a capture membrane. The collector membrane contains an antibody against a first epitope of the protein to be determined and the capture membrane contains an antibody against a second epitope of the protein to be determined.
EPO 0420 021 A2 discloses a method for determining the presence or an amount of a ligand-analyte in a sample using a porous, wettable membrane solid phase having an immobilized ligand receptor capable of binding the ligand, wherein the membrane is laminated to a support using a water solvent based adhesive. Membranes laminated to the support by this method include nitrocellulose and polyvinylidene difluoride (PVDF) membranes.
WO 95/06252 discloses a method for identifying an immunological substance using a multimembrane system which comprises distributing an aqueous mixture of a liquid sample believed to contain the immunological substance and a conjugate thereof coupled to a marker over a selective phase consisting of a porous membrane including an immobilized immunological reagent which either reacts with the conjugate and conjugate coupled to the marker but not the conjugate when bound to the immunological substance or reacts with the conjugate when bound to the immunological substance but not the conjugate alone or conjugate coupled to the marker.
Other attempts to increase sensitivity of ELISAs include increasing the binding capacity of the support. Several such attempts have been directed to changing the chemical configuration of the surface so that it will form a chemical bond with the ligand. For example, U.S. Pat. No. 4,933,410 discloses activating polystyrene supports by reacting the surface of the support with hydroxylmethylamides in a polystyrene insoluble solvent. Similarly, U.S. Pat. No. 4,119,589 discloses activating a compound having at least two secondary amine groups by converting the secondary amine groups into imino-chloride groups. Methods of increasing binding capacity of a solid support by first coating the support with an inert protein which binds to the ligand by adsorption, ionic binding, entrapment or covalent binding have also been described in U.S. Pat. No. 4,210,418.
Methods of increasing both the capacity and affinity of a polymeric solid support by activation of the solid support by solvent treatment or mechanical means such as grinding or sanding are described in the U.S. Pat. No. 5,424,219.
A method of increasing the specificity of an ELISA assay for a selected antibody, namely antibodies specific to antiphospholipid syndrome, wherein a negative charge or a lone pair of electrons and/or free radicals containing a negative charge or a lone pair of electrons are introduced via electron beam is described in U.S. Pat. No. 5,472,883.
Accordingly, there remains a need for solid support-based immunoassays which produce reliable and quick results.